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Cloning, sequencing, and overexpression in Escherichia coli of the alpha-D-glucose-1-phosphate cytidylyltransferase gene isolated from Yersinia pseudotuberculosis.

ABSTRACT: A clone of Yersinia pseudotuberculosis DNA carrying the ascA gene was constructed, and the corresponding protein was successfully overexpressed in Escherichia coli. A protocol consisting of DEAE-cellulose and Sephadex G-100 column chromatography was developed and led to a nearly homogeneous purification of the ascA product. Initial characterization showed that the ascA-encoded protein is actually the alpha-D-glucose-1-phosphate cytidylyltransferase which catalyzes the first step of the biosynthesis of CDP-ascarylose (CDP-3,6-dideoxy-L-arabino-hexose), converting alpha-D-glucose-1-phosphate to CDP-D-glucose. In contrast to early studies suggesting that this enzyme was a monomeric protein of 111 kDa, the purified cytidylyltransferase from Y. pseudotuberculosis was found to consist of four identical subunits, each with a molecular mass of 29 kDa. This assignment is supported by the fact that the ascA gene, as a part of the ascarylose biosynthetic cluster, exhibits high sequence homology with other nucleotidylyltransferases, and its product shows high cytidylyltransferase activity. Subsequent amino acid comparison with other known nucleotidylyltransferases has allowed a definition of the important active-site residues within this essential catalyst. These comparisons have also afforded the inclusion of the cytidylyltransferase into the mechanistic convergence displayed by this fundamental class of enzyme.

SUBMITTER: Thorson JS

PROVIDER: S-EPMC205285 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

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Cloning, sequencing, and overexpression in Escherichia coli of the alpha-D-glucose-1-phosphate cytidylyltransferase gene isolated from Yersinia pseudotuberculosis.

Thorson J S JS Kelly T M TM Liu H W HW

Journal of bacteriology 19940401 7

A clone of Yersinia pseudotuberculosis DNA carrying the ascA gene was constructed, and the corresponding protein was successfully overexpressed in Escherichia coli. A protocol consisting of DEAE-cellulose and Sephadex G-100 column chromatography was developed and led to a nearly homogeneous purification of the ascA product. Initial characterization showed that the ascA-encoded protein is actually the alpha-D-glucose-1-phosphate cytidylyltransferase which catalyzes the first step of the biosynthe ...[more]

PMID: 8144449

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Project description:BackgroundThe production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes.ResultsA set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities.ConclusionsThe use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.

Dataset Information

Cloning, sequencing, and overexpression in Escherichia coli of the alpha-D-glucose-1-phosphate cytidylyltransferase gene isolated from Yersinia pseudotuberculosis.

Publications

Cloning, sequencing, and overexpression in Escherichia coli of the alpha-D-glucose-1-phosphate cytidylyltransferase gene isolated from Yersinia pseudotuberculosis.

Similar Datasets

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